This Article Abstract Full Text (PDF) All Versions of this Article: 21/1/1    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager NURSA Molecule Pages Link Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harris, H. A. Search for Related Content PubMed PubMed Citation Articles by Harris, H. A.

Estrogen Receptor-ß: Recent Lessons from in Vivo Studies

Women’s Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Heather Harris, Women’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, RN3163, Collegeville, Pennsylvania 19426. E-mail: harrish{at}wyeth.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
The unexpected discovery of a second form of the estrogen receptor (ER), designated ERß, surprised and energized the field of estrogen research. In the 9 yr since its identification, the remarkable efforts from academic and industrial scientists of many disciplines have made significant progress in elucidating its biology. A powerful battery of tools, including knockout mice as well as a panel of receptor-selective agonists, has allowed an investigation into the role of ERß. To date, in vivo efficacy studies are limited to rodents. Current data indicate that ERß plays a minor role in mediating estrogen action in the uterus, on the hypothalamus/pituitary, the skeleton, and other classic estrogen target tissues. However, a clear role for ERß has been established in the ovary, cardiovascular system, and brain as well as in several animal models of inflammation including arthritis, endometriosis, inflammatory bowel disease, and sepsis. The next phase of research will focus on elucidating, at a molecular level, how ERß exerts these diverse effects and exploring the clinical utility of ERß-selective agonists.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
ESTROGENS, LIKE MANY other hormones, elicit a myriad of biological responses, and it has been appreciated since the early 1960s that these actions are receptor mediated. When an estrogen receptor (ER) was cloned in 1986 (1, 2) and its ablation in mice had the expected phenotype in many organ systems (3), it seemed a plausible conclusion that only one ER existed. However in 1996, Jan-Ake Gustafsson announced at a Keystone Symposium that his group had discovered a second form of the ER, necessitating the first cloned receptor be renamed ER. This second receptor, designated ERß (4), was unexpectedly found while searching for additional androgen receptors using a rat prostate cDNA library and degenerate PCR primers. It would not be an overstatement to say that this discovery electrified the field of estrogen biology, causing a reexamination of all our understandings and assumptions about the mechanism of estrogen action.

Early studies, naturally, focused on cloning ERß from additional species (5, 6) and describing ERß’s ligand specificity (7, 8, 9) and tissue distribution (10, 11, 12, 13). Two key conclusions can be drawn from this body of work: First, 17ß-estradiol is the most potent endogenous ligand for ERß, and it binds equally well to ER and ERß. Second, ERß is widely distributed, although it is not highly expressed in the uterus. One logical extrapolation of this information is that a selective ERß ligand would be expected to mimic estradiol’s actions in a variety of target tissues but would have less impact on the uterus. This was the premise behind part of the drive to identify/design selective ligands: not only could these compounds help dissect ER from ERß function but they might also carry clinical benefits. Early speculation about potential clinical benefits was primarily related to the treatment of menopausal disorders such as osteoporosis, cardiovascular disease, cognition, and urinary incontinence (14). If this list seems expansive and unduly ambitious, recall that ERß’s discovery came at a time when estrogen therapy in postmenopausal women was thought (at least at a research level) to potentially confer benefits on almost all organ systems and was prescribed clinically for long-term use.

This article will review the current state of knowledge about the function of ERß and will be biased toward discussion of recent in vivo data generated from ER and ERß knockout (KO) mice as well as a summary of the preclinical characterization of ER- and ERß-selective agonists. A number of other reviews on ERß have recently been published that examine estrogen action in general and lessons learned from in vitro studies (15, 16, 17).

	SUMMARY OF AVAILABLE TOOLS AND CAVEATS

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
ERKO Mice
Mice lacking both ER subtypes have been produced. Two versions of ERKO mice exist. The first mouse constructed (Ref. 3 ; designated ERKO_CH in this review) is still the most widely used, but residual effects of estrogens in this animal cannot be automatically attributed to ERß because of low levels of ER splice variants that retain some activity (18). More recently, an apparently complete KO of ER has been accomplished, and these mice will be designated ERKO_ST (19).

Three groups have independently constructed mice lacking ERß, and there is disagreement in the field as to their phenotype. In this article, ßERKO mice will be designated as follows: ßERKO_CH for the mice produced in Chapel Hill by Krege et al. (20) that are commercially available from Taconic Farms, Inc. (Germantown, NY); ßERKO_KI for the colony of these mice that were subsequently established at the Karolinska Institute; ßERKO_ST for the mice produced by Dupont et al. (19) in Strasbourg, France; and ßERKO_WYE for the mice produced by Wyeth (21).

Although independently generated KO mice might not be expected to exhibit identical phenotypes, the issue confronting scientists studying ßERKO mice is unexpected in that investigators studying ßERKO_CH and ßERKO_KI observe markedly different phenotypes (detailed examples discussed below), whereas the ßERKO_ST and ßERKO_WYE mice seem similar to the ßERKO_CH mice. Potential explanations for the phenotype differences have been discussed but not rigorously tested. One hypothesis is that factors present in the ßERKO_KI environment elicit the phenotypes seen. These might include dietary differences or the presence of endemic pathogens. This hypothesis is readily testable by cohousing ßERKO_CH mice with ßERKO_KI mice in the Karolinska Institute’s colony, but this has not been done as yet. Although the data generated from the ßERKO_KI mice are dramatic and provide compelling evidence for a role for ERß in a variety of diseases, it is very puzzling that similar observations have not been made in other ßERKO mice. As with the selective compound data discussed below, and indeed with any scientific observation, the dataset inspiring the highest confidence is obtained from independently corroborating observations.

Selective Small Molecules
A number of selective ERß agonists have been described in the peer-reviewed scientific literature (see Ref. 22 for a comprehensive review including patent sources), but a minority has been evaluated extensively in vivo. These include DPN (23), ERB-041 (24), WAY-202196 (25), WAY-200070 (24), and 8ß-VE₂ (26) (Fig. 1). Selectivity of these compounds was measured in in vitro screens using transcriptional assays and/or competitive radioligand-binding assays. All of these compounds are at least 70-fold selective for ERß over ER (based on a radioligand-binding assay), and their potencies range from 18–180% that of 17ß-estradiol (Table 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Structures of Selective ER Agonists

Although good comparative data exist for both types of ER-selective compounds on classic estrogenic endpoints and the results are, by-and-large, consistent (see below), relatively few studies have been published on the ERß agonists’ positive in vivo activity, and few direct comparisons have been made. Therefore, it is premature to make many statements about ERß class effects that will be shared among all members. Just as selective ER modulators can vary in their efficacy on a given target, it is to be expected that even bona fide selective ERß agonists will exhibit a range of activities. Moreover, typically these ERß-selective compounds were classified as agonists based on their profile in (an often) contrived in vitro model or using three-dimensional x-ray cocrystal structure. It is entirely possible that the compounds described to date do not elicit the full range of biological activities able to be exhibited by ERß. Thus, the accurate definition of ERß-mediated activities requires the simultaneous testing of a wide range of highly selective ligands in a variety of in vivo models.

	ER MEDIATES THE MAJORITY OF ESTROGEN’S EFFECTS ON CLASSIC ESTROGEN TARGET TISSUES

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
Estrogens that lack marked selectivity for either ER, such as 17ß-estradiol or 17-ethinyl-17ß-estradiol, have well-characterized and familiar effects on several organ systems. These include stimulation of the uterus, facilitation of proliferation/endbud development in the mammary gland, control of ovulation, maintenance of bone mineral density, and negative feedback to the hypothalamus and pituitary. As has been reviewed extensively elsewhere (16, 31), mice not expressing ER lack many of these responses. Although the published literature is not unanimous on the subject, on balance, the data suggest that ERß plays a minor role in several organs/organ systems that are highly responsive to estrogens. These are discussed in more detail below.

Uterus
ERKO_CH mice have well-documented defects in uterine responses to estrogens (reviewed in Ref. 31). However, nonselective estrogens cause an equivalent increase in uterine wet weight in wild-type and ßERKO_CH and ßERKO_WYE mice (Korach, K. S., and H. A. Harris, unpublished observations). Moreover, microarray studies indicate that both the early and late genomic responses to estradiol are indistinguishable between wild-type and ßERKO_CH mice (32). In contrast to these findings, the ßERKO_KI mice exhibit an enhanced response to estrogenic stimulation as measured by luminal fluid production and expression of complement C3 protein (33).

The data gathered from selective ER agonists are consistent with the KO mouse phenotype, suggesting a minor role for ERß in uterine physiology. ER appears to be necessary and sufficient to mediate estrogen’s increase in wet weight (at least in rats) as evidenced by the fact that PPT (28) and 16-LE₂ (26) are as efficacious as reference estrogens on this endpoint. In terms of ERß-selective compound activity, uterine weight has been measured for all five selective agonists. DPN, ERB-041, WAY-202196, and WAY-200070 do not significantly increase uterine weight at 50 mg/kg when given sc to rats and/or mice (Refs. 24 and 25 ; and Harris, H. A., unpublished observations). The steroidal selective agonist, 8ß-VE₂, increased uterine weight at a sc dose of 0.5 mg/kg. It is interesting to note that the in vivo selectivity of 8ß-VE₂ is more in line with expectations based on its in vitro selectivity than the other compounds, which appear more selective in vivo that what would be predicted from their in vitro potency and selectivity. There are potential explanations for this observation. First, there is not an exact relationship between binding and activity. Second, these compounds are likely differentially bound to plasma proteins and/or metabolized.

Mammary Gland
Data from ERKO_CH, ßERKO_CH, and transgenic aromatase/ERKO_CH mice clearly indicate that ERß is not required for normal mouse mammary gland development or function (16, 34). In contrast, ßERKO_KI mice have enlarged alveoli and reduced expression of several markers of differentiation and a concomitant increase in the proliferation marker Ki67 (35). Little published data currently exist on the effects of selective ER agonists on estrogen-dependent functions in the mammary gland.

Hypothalamic/Pituitary Effects
In contrast to ERKO_CH mice, ßERKO_CH mice have normal circulating levels of LH and estradiol (36), suggesting that ER controls the negative feedback of estrogens to the brain (16). Similar to estradiol, 16-LE₂ suppressed LH and FSH production in the ovariectomized rat, again showing ER sufficiency in mediating this negative feedback loop. When administered at nonuterotrophic doses, the ERß-selective agonists DPN, WAY-200070, and 8ß-VE₂ had no effect on FSH and/or LH nor did they inhibit ovulation, suggesting the inability of ERß to influence this axis (26, 29, 37, 38).

Another well-known effect of estrogens on the pituitary is the stimulation of prolactin secretion. In mice, PPT increased prolactin levels whereas DPN had no effect. It should be noted that the efficacy of PPT was less than that of estradiol at the dose tested, but that a full dose-response study was not performed (39).

Skeleton
Careful characterization of ER null mice has shown a variety of phenotypes, suggesting that both ER and ERß play a role in bone development. However, these effects are not seen at all ages in both genders or all investigators’ ßERKO mice (see Ref. 36 for review). Moreover, estradiol protected against ovariectomy-induced bone loss in ßERKO mice, indicating that ERß is not necessary for this activity. Extrapolation of these findings in rodents to effects in humans is problematic; however, an unambiguous role was established for the necessity of ER in normal bone development when a human male was discovered with an inactivating mutation in this receptor (40). This individual had unfused epiphyses and markedly reduced bone mineral density and was unresponsive to estradiol therapy.

The activity of selective ERß agonists supports the idea that ERß is not a major player in estradiol’s maintenance of adult skeletal mass. As with the uterine responses, selective ER stimulation with PPT or 16-LE₂ is as efficacious as a reference estrogen at preservation of bone mineral density after ovariectomy in rats (26, 28), whereas ERB-041, WAY-200070 or 8ß-VE₂ were inactive at nonuterotrophic doses (24, 26, 41).

Ovarian Physiology
ERß is highly expressed in the granulosa cell, and all types of ßERKO mice are subfertile. Ovaries from both ßERKO_CH and ßERKO_ST mice have fewer corpa lutea than their wild-type counterparts but only the ßERKO_CH mice showed an increase in the number of atretic follicles (19, 20). Under gonadotropin stimulation, ßERKO_CH and ßERKO_ST mice produced fewer oocytes, although the severity of impairment varied and likely represented partial compensation in some animals by ER. These data indicate a role for ERß in folliculogenesis and are supported by the activity of 8ß-VE₂ in the hypophysectomized rat (29).

Potential for ERß-Selective Compounds to Block Estradiol’s Activity
Given the inactivity of selective ERß agonists in classic estrogen target tissues, several studies have examined the potential for these compounds to block a reference estrogen’s effects. In the uterus, when WAY-200070, ERB-041, or 8ß-VE₂ are coadministered with 17ß-estradiol, these compounds do not block the estradiol-mediated increase in uterine weight (24, 26, 41). Additionally, WAY-200070 did not block the antiresorptive effects of 17ß-estradiol in rat models of osteopenia (24), and 8ß-VE₂ was unable to block 17ß-estradiol’s effects on pituitary (gonadotropins) or liver (angiotensin I, IGF-I, high density lipoprotein, or total cholesterol) responses (26). DPN did not antagonize PPT effects on LH, FSH, or prolactin (37).

Frasor et al. (39) have found that DPN has the potential to mildly antagonize the uterine effects of PPT. In the mouse, coadministration of DPN with a modest dose of PPT led to a 30% reduction of PPT-induced wet weight. Additionally, mild inhibitory effects were seen on some mRNA markers of estrogen action in the uterus. On the other hand, DPN had modest stimulatory effects on the activity of PPT on other markers of estrogen action. It remains to be determined whether these effects indicate a subtle modulatory effect of ERß on uterine response to estrogens or if they are observations specific to DPN.

	ROLES FOR ERß IN NONREPRODUCTIVE ORGAN SYSTEMS

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
ERß’s activity has been investigated in a number of in vivo models, some using KO mice and others using selective agonists. Major areas of investigation are presented below, grouped roughly by organ system or disease type.

Prostatic Actions
The role of estrogens in prostatic physiology is controversial. Historically, high doses of estrogens were used to treat prostate cancer, but it was thought to act via down-regulation of gonadotropins. When ERß was discovered in the prostate epithelium, it stimulated investigation of direct interactions of estrogens on the prostate. Although neonatal exposure to high amounts of estrogens alters prostate development, two studies using KO mice (ßERKO_KI, ßERKO_CH, and ERKO_CH) clearly show this imprinting is mediated by ER (42, 43).

Older ßERKO_KI mice have hyperplastic prostates (44), a phenotype that is not seen in ßERKO_CH (42, 45) or ßERKO_ST (19) mice. In addition to abnormal histological changes in these mice, markers of proliferation are increased and androgen receptor (protein) expression is increased. It appears that the prostate locally produces a dihydrotestosterone metabolite that is ligand for both ERs: 5-androstane-3ß,17ß-diol (44). Exogenous administration of this compound to mice decreased markers of cell proliferation in the developing prostate of wild-type mice and this effect was absent in ßERKO_KI mice (46). However, as pointed out by Taylor et al. (47), because the prostate is so androgen sensitive, it is difficult to separate direct effects of estrogens on this organ from their ability to modulate androgen action. Accordingly, this group used organ cultures of prostates isolated from rats (newborn) or wild-type ERKO_CH, and ßERKO_CH mice (postnatal d 1). In cultures of both rat and mouse prostates, testosterone supports growth and branching, whereas coadministration of estradiol reduced growth and promoted apoptosis, especially in the distal epithelial cells (areas of active branching). These proapoptotic effects were seen in both ERKO_CH and ßERKO_CH mice, suggesting neither ER is involved. Because double KO mice were not available, selective agonists were used to try and mimic estradiol’s effects. However, neither PPT, DPN, nor 5-androstane-3ß,17ß-diol could replicate the effects of estradiol on the explants, although they each had other effects on prostate morphology. It should be noted that although low levels of testosterone were required to maintain tissue development, very high concentrations of estradiol were used to see the proapoptotic effects (15 µM). These concentrations are similar to those used for neonatal imprinting studies but may have little relevance to adult exposures. Further analysis of the estradiol-treated prostates showed that ERß protein was up-regulated, and androgen receptor protein was down-regulated in distal areas of prostates, regardless of genotype. Thus estrogens, especially at pharmacological doses in the developing organ, may impact prostate physiology independent of either ER.

Vasomotor Instability
Estrogens are frequently prescribed for the relief of hot flushes in peri- or postmenopausal women. Both ERs are expressed in the hypothalamus, the thermoregulatory center of the brain; therefore it is possible that estrogen’s suppression of vasomotor instability could be mediated by ERß and/or ER. This question has been investigated using both KO mice and selective ER agonists, and studies agree that activation of ER is sufficient to modulate tail skin temperature in the rodent, an animal model of hot flush. First, PPT was as effective as ethinyl estradiol at reducing tail skin temperature changes induced by acute morphine withdrawal in ovariectomized rats (28). Second, Opas et al. (48) found that estradiol cypionate was as effective at suppressing ovariectomy-induced tail skin temperature increase in ßERKO_CH as wild-type mice. The data on whether ERß activation has a similar effect are mixed. In the Opas et al. study mentioned above, estradiol cypionate also worked well in ovariectomized ERKO_CH mice, whereas WAY-200070 was inactive in the morphine model (24). Additional studies are needed to resolve this apparent inconsistency as to the role of ERß in mediating estrogen’s actions on hot flushes.

Cardiovascular System
Estrogens have well-appreciated effects on the cardiovascular system in preclinical models, but human clinical trial data have not been unanimously supportive. Reasons behind this apparent discrepancy are under investigation (see Ref. 49 for review) and may depend on the age at which treatment is given (50). Despite this controversy, enthusiasm for defining the role of the ER subtypes in mediating estrogen’s preclinical effects remains high. KO mice and selective ER agonists have been used to investigate the relative contributions of ERß and ER in models of vascular injury, vascular function, and cardiac injury.

After carotid artery denudation, vascular smooth muscle cells proliferate, leading to a narrowing of the vessel lumen. Estradiol can prevent this neointima formation in both rats and mice. Early studies using ERKO_CH mice suggested estradiol might work via ER or ERß to prevent intimal thickening, but more recent data from ERKO_ST mice suggest ER is the sole receptor responsible (51). No highly subtype-selective agonists have been tested in this model; thus it remains to be seen whether these tools will confirm observations from the KO mice.

Several studies have examined ER subtype effects on vascular function. Estradiol does not reduce phenylephrine-induced contraction in endothelial-denuded aortic vascular rings of ßERKO_CH mice as it does in wild-type mice, and this defect is due to reduced inducible nitric oxide synthase produced (52). In keeping with these results, rat aortic rings with intact endothelia precontracted with noradrenaline relaxed significantly when treated acutely with nanomolar levels of estradiol or PPT, whereas DPN had no significant effect over a concentration range expected to be selective for ERß (53). The PPT effect was blocked by ICI-182780, strongly suggesting an ER-dependent effect. These data suggest ER mediates relaxation effects but is in contrast to the work reported by others (54, 55) that use PPT and DPN to support a role for both ERs. However, these other studies used very high (>1 µM) concentrations of compounds that are clearly pharmacological and beyond the range where these compounds are ER or ERß selective in in vitro assays (23, 27). A study in the spontaneously hypertensive rat confirms an obligatory role for ER in mediating vessel relaxation (56). In this study, ovariectomized rats were treated for 4 wk, after which aortic rings were isolated for testing. Aortic tissue from rats treated with estradiol or 16-LE₂ (Cpd1471) and precontracted with KCl was more responsive to acetylcholine-induced relaxation than tissue from ovariectomized rats. These effects were mediated by the endothelium, as contraction/relaxation profiles of denuded aortic rings were not different between the treatment groups. Moreover, endothelial nitric oxide synthase was up-regulated by treatment with estradiol and 16-LE₂, again pointing to an ER-dependent effect on the endothelium.

Three studies have looked at in vivo effects of ER subtypes in models of cardiac injury. Korte et al. (57) looked at the effect of ER or ERß deletion on various cardiac functions after anterior myocardial infarction. Genotype did not influence mortality or infarction size [in contrast to the findings of Pelzer (58) discussed below]. Moreover, the ERKO_ST mice had no defects in ECG parameters. In contrast, the ßERKO_CH mice had prolonged QT, QTc, JT, and JTc after infarction when compared with wild-type mice (regardless of injury) and sham-operated ßERKO_CH controls. Moreover, there was a dramatic ERß-dependent reduction in ventricular premature beats. These phenomena may be attributed to decreased expression of the potassium channel Kv4.3 mRNA seen in the ßERKO_CH mice.

In a similar study of chronic anterior myocardial infarction, ßERKO_CH mice had lower survival rates, retained more fluid, and had elevated levels of ventricular pro-atrial naturetic peptide than their wild-type counterparts. Thus, although the data are not entirely concordant, both studies conclude that ERß plays a role in the response to myocardial infarction. Future studies will likely test whether selective ER agonists are protective in this model.

Finally, in an ex vivo ischemia/reperfusion model using hearts excised from ERKO_CH and ßERKO_CH mice and treated with isoproterenol, Gabel et al. (59) found that female hearts sustained less injury (as measured by a reduction in infarction size) than males, and that this was attributable to ERß. Expression profiling suggested that ßERKO_CH females had reduced levels of various genes involved in fatty acid metabolism, thus perhaps providing a mechanistic explanation.

When the heart is subjected to increased pressure via aortic constriction, cardiac hypertrophy develops. Skavdahl et al. (60) observed that male mice developed larger hearts than females and further discovered, using ERKO_CH and ßERKO_CH mice, that ERß was responsible for this attenuation. However, a study in spontaneously hypertensive rats indicates that stimulation of ER is protective (30). These rats develop cardiac hypertrophy as a consequence of chronically elevated blood pressure. Although estradiol or 16-LE₂ treatment for 12 wk did not lower blood pressure, both compounds reduced heart weight and improved cardiac function. These effects were blocked by ICI-182780, indicating the activity depends on an ER. It remains to be seen whether ERß agonists can also achieve this effect. It will also be interesting to determine the effect of selective ERß agonists on blood pressure because the ßERKO_CH mice have hypertension (52).

Brain and Behavior
ERß is expressed throughout the brain (see Ref. 10 for review), and there has been much interest in defining its function. Specific areas of investigation have been stroke/neuroprotection, anxiety/depression, and learning/memory.

Estrogens protect against neuronal cell loss in a variety of ischemic stroke models. These models differ in whether the tissue is reperfused and the level of estradiol required for protection; thus the data are not entirely comparable. Early data using ßERKO_WYE and ERKO_CH mice indicated an obligatory role for ER (61) in preserving neurons after middle cerebral artery occlusion leading to permanent cerebral ischemia. More recently, using a 15-min global ischemic insult, followed by reperfusion, DPN significantly reduced neuronal damage in the caudate nucleus and CA1 pyramidal cell layer (62) in mice. A similar study by Miller et al. (38) found both WAY-200070 and PPT protected the CA1 region of the hippocampus although there was a pronounced bimodal distribution among the animals within a group.

One area of particularly active investigation is whether ERß might ameliorate anxiety and/or depression. Support for this hypothesis is derived from distribution studies as well as estradiol/ERß influences on the serotonergic system. Double immunolabeling studies show that more than 90% of ERß-expressing neurons also express tryptophan hydroxylase, the rate-limiting step in serotonin synthesis; although deletion of ERß in ßERKO_CH mice does not affect its expression (63). A functional connection between these two proteins was made by the discovery that estradiol up-regulates tryptophan hydroxylase-1 mRNA via ERß in the dorsal raphe nucleus and shown by comparing estradiol’s activity in wild-type and ßERKO_CH mice (64). Interestingly, such regulation may have a local effect in the midbrain, because estradiol does not affect tryptophan hydroxylase-2, the more widely expressed isoform. It remains to be shown whether this increase in mRNA translates to increased protein expression.

A number of behavioral studies have been published that use either KO mice or selective ER agonists to investigate the effects of ERß on anxiety and/or depression. In the forced swim test model of depression using mice, estradiol reduced immobility time comparably to a reference compound, desiprimine, and estradiol’s effect (but not that of desiprimine) was lost in ßERKO_CH mice (65). ERKO mice have not been evaluated as yet. Rats’ performance in the forced swim test also implicates a role for ERß: immobility times were similarly reduced when rats were given estradiol, DPN, and desiprimine, but PPT had no effect (66).

In a model measuring anxiety, Krezel et al. (67) reported an increase in anxiety-related behaviors of ßERKO_ST female mice in the elevated plus maze or in the open-field test, perhaps attributable to abnormalities in the amygdala. These behavioral observations were confirmed by Inwalle et al. (68) using ßERKO_CH mice, and it was further reported that estradiol did not influence the behavior of either genotype. Two other studies used rats and measured the time they spent in a lighted open field or the open arm of an elevated plus maze (69, 70). Estradiol and DPN clearly increased time spent in the open area, and PPT had no effect although it affected other behaviors. Moreover, these antianxiety effects of estradiol and DPN were effectively blocked by coadministration of tamoxifen, suggesting an ER-mediated effect.

ERß’s role in synaptic plasticity and learning is also being investigated (71). Male and female wild-type and ßERKO_WYE mice were trained to associate a shock with a particular context (operant chamber) and cue (tone). Later, mice were tested to assess the memory strength for the context-shock relationship by placing them in the same chamber, but without sounding the cue. In this situation, wild-type mice freeze because they recognize the surroundings and associate them with the electric shock. Both male and female ßERKO_WYE mice were significantly impaired in their freezing response to the context—indicative of failed associative learning of the shock with the context. It is thought that that memory encoding and retrieval of a context is dependent on the hippocampus. It is well established that genetic manipulations that disrupt hippocampal memory also disrupt forms of hippocampal synaptic plasticity, such as long-term potentiation. Interestingly, hippocampal slices taken from female ßERKO_WYE exhibit a robust long-term potentiation deficit in area CA1 compared with wild-type mice. Specifically, fast synaptic transmission was impaired and long-term potentiation responses were blunted, and both of these deficits would be expected to impact learning and memory.

Models of Inflammatory Bowel Disease and Arthritis
Nonselective estrogens are known to have antiinflammatory properties and to interfere with NFB activity (72, 73) and thus it was possible that ERß might be capable of mediating this activity. Many models of inflammation exist, but the first model we used was the HLA-B27 transgenic rat. These rats develop inflammation in the intestine, joint, and skin and are used as models of inflammatory bowel disease and rheumatoid arthritis (74). When administered to fully diseased rats, ERB-041 and WAY-202196 reversed the animals’ symptoms of chronic diarrhea and reduced intestinal inflammation (25, 41). Coadministration of ICI-182780 completely blocked the effect of ERB-041 on stool character, providing strong evidence that its effect was ER dependent. At least seven other ERß-selective agonists, representing several different chemical series, also have this effect (Ref. 24 ; and Harris, H. A., and J. C. Keith, Jr., unpublished observations). In addition, ERB-041 and WAY-202196 prevent or reverse established arthropathy in these rats as reflected by a reduction in the clinical signs of joint redness/swelling as well as synovitis and Mankin histological scores (Refs. 24 and 25 ; and Harris, H. A., and J. C. Keith, Jr., unpublished observations).

ERB-041 and WAY-202196 were also tested in the Lewis rat adjuvant-induced arthritis model, a well-established model of rheumatoid arthritis. Both these compounds reduced clinical signs of arthritis to near normal levels and significantly improved joint histology scores. In addition, ERB-041 reversed the majority of gene expression changes caused by disease induction in the liver, lymph node, and spleen and, as well, reversed many of the disease-induced plasma proteome changes (75).

Although nonselective estrogens/selective ER modulators are active in both these models (76, 77), ER-selective compounds have not been tested. Moreover, these observations cannot be confirmed using ERKO mice because they rely on specific genetic models of disease. Our experience with Wyeth ERß-selective agonists is that they are not general antiinflammatory agents because they are inactive in many other models of inflammation, including collagen-induced arthritis, dextran sulfate sodium-induced colitis, carrageen-induced paw edema, lipopolysaccharide-induced haptoglobin production, experimental allergic encephalitis, and passive cutaneous anaphalaxis (Ref. 78 and Harris, H.A., unpublished observations). Finally it should be mentioned that a clear role for ER-dependent antiinflammatory activity has been established in some in vivo models; thus, antiinflammatory activity is not a unique property to ERß (78, 79, 80, 81).

Endometriosis
Endometriosis is an estrogen-dependent disease with a significant inflammatory component (82). Although it may seem counterintuitive to evaluate an ER agonist in models of this disease, given the antiinflammatory activity seen in other models, it seemed a reasonable idea to pursue. Although this disease occurs naturally only in primates, several rodent models exist, and one well-accepted model uses human endometrial tissue implanted as xenografts into nude mice (83, 84). ERB-041 was tested in both ovariectomized and gonad intact mice with established endometriosis lesions, and after 2 wk of treatment, 40–75% of the mice were completely lesion free (85). Only ER was detected in recovered lesions (regardless of treatment), and thus it is speculated that ERB-041 may be stimulating the macrophages and/or natural killer cells of the nude mice to recognize the exogenous endometrial tissue as foreign and clear it. Clearly the in vivo profile of a selective ERß agonist is not only a subset of estradiol’s activity.

Trauma and Sepsis Models
It has become apparent in recent years that females have a lower susceptibility for, and an improved survival from, traumatic injury and sepsis (86). There is a large body of work showing androgen’s deleterious effects and estrogen’s protective qualities. Recent work is beginning to delineate the contributions of ER and ERß to this beneficial effect. For the sake of brevity, only data relating to ERß will be presented here. In a rat model of trauma-hemorrhage, which examines lung injury, estradiol and DPN were equally effective in reducing a variety of inflammatory mediators including inducible nitric oxide synthase and IL-6, as well as markers of tissue damage such as lactate dehydrogenase and total protein content of the bronchioalveolar fluid. PPT was ineffective on these parameters (87). In a similar study, designed to examine cardiac function, DPN, but not PPT, improved cardiac output and stroke volume. In addition DPN, but not PPT, maintained heat shock proteins 32, 60, 70, and 90 (88).

WAY-202196 has been examined in three models of systemic infection, the second and third of which are used as models of sepsis: 1) mouse listeriosis, 2) Pseudomonas infection in the neutropenic rat, and 3) mouse cecal ligation and puncture (89). Whereas a single oral dose of WAY-202196 did not positively or negatively affect response to Listeria infection, a monoclonal antibody to TNF increased susceptibility to infection (lowered the LD₅₀). Thus, WAY-202196 was not globally immunosuppressive in this model.

The Pseudomonas infection model aims to replicate the human situation of an immunocompromised patient (e.g. cancer patient receiving radio- or chemotherapy) who develops an infection. In this model, multiple doses of WAY-202196 increased survival (25–83%) and improved intestinal histology. The mouse cecal ligation and puncture model induces acute bacterial peritonitis (for review, see Ref. 90). When WAY-202196 was tested in this model, it improved survival (0% vs. 83%) and also reduced intestinal injury.

Leukemia
At 18 months of age, ßERKO_KI mice of both sexes have grossly enlarged spleens compared with their wild-type counterparts, and is thought to be a consequence of bone marrow hypercellularity (91). Moreover, these mice develop neoplasia resembling human chronic myeloid leukemia. To date, this phenotype has not been reported in other strains of ßERKO mice.

	UNANSWERED QUESTIONS ABOUT ERß

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
Is There a Unifying Mechanism of Action for ERß-Mediated Activities?
As outlined above, ERß potentially affects many organ systems, and selective compounds have the potential to treat several disparate diseases. The field is currently sorely lacking in mechanistic hypotheses to explain how ERß might exert these effects. Although this dearth is understandable, not only because ERß was recently discovered, and within precedent, because we have no true mechanistic understanding of how estrogens work via ER to ameliorate hot flushes and bone loss (despite having known about these activities for decades), mechanistic uncertainty undoubtedly handicaps further research. It seems doubtful that it will be possible to explain the diverse effects of this transcription factor with a single mechanism. Having said that, however, one system that bears further investigation is the hypothalamic-pituitary-adrenal (HPA) axis. Modulation of this axis may explain effects in certain inflammatory and behavioral models.

First, looking at expression in relevant brain regions, ERß is the predominant ER expressed in the rat paraventricular nucleus (10), which is a key mediator of the stress response. Additionally, there is evidence for an interplay between corticosterone, estradiol, and ERß expression in the paraventricular nucleus (92). Finally, ERß is colocalized with a subset of CRH neurons in the rat paraventricular nucleus and in vitro, ERß can up-regulate the CRH promoter in response to estradiol (93).

Several studies demonstrate an effect of nonselective estrogens on the HPA. It should be noted that effectiveness on these endpoints is highly dependent on dose and regimen. For the sake of brevity, only a few studies will be mentioned here. A single episode of restraint stress markedly elevated ACTH and corticosterone levels in rats, but chronic treatment with estradiol benzoate significantly reduced the ACTH response (with no effect on corticosterone levels) (94). Interestingly, in this study, estradiol benzoate increased levels of other stress-responsive markers (adrenal tyrosine hydroxylase and dopamine ß-hydroxylase mRNA) in unstressed animals, pointing out the system’s complexity. In another stress-dependent model, estradiol reduced stress-induced expression of c-Fos in the paraventricular nucleus, although (as above) corticosterone levels were unaffected (95).

Other studies have measured behavioral consequences of HPA modulation. In a study discussed above, Lund et al. (70) showed that all rats tested in the elevated plus maze had increased levels of corticosterone, but the levels seen in the DPN-treated animals were significantly lower than those treated with estradiol or PPT (which were significantly higher than vehicle-treated rats in contrast to the findings of Ref. 96). Because both estradiol and DPN were effective at reducing anxiety in this model, the effects would seem not explainable by modulation of corticosterone levels, although it should be noted that it was only measured at one time point (30 min after returning to home cage).

In a similar study again using the open field and elevated plus maze tests, Walf and Frye (96) found that estradiol’s effects in reducing anxiety were blunted by pretest restraint stress. Doses of estradiol that were effective in reducing stress also reduced corticosterone levels and these effects were also seen in unstressed untested rats. Estradiol’s antianxiety action was likely not entirely via modulation of corticosterone levels, because although estradiol was ineffective in adrenalectomized rats, this was reversed by administration of low doses (but not high doses) of corticosterone.

Lewis rats, which are used for the adjuvant-induced arthritis model, have well-known defects in their HPA axis that render them hypersensitive to inflammatory stimuli (97). Lipopolysaccharide stimulation of Fischer 344 rats (the control strain for Lewis rats) leads to robust up-regulation of CRH mRNA in the paraventricular nucleus and a modest down-regulation of ER and ERß mRNA (97). Neither of these responses is seen in Lewis rats, again suggesting interplay between ERs and the HPA.

Are There Expected Clinical Liabilities with Selective ERß Agonists?
Based on the biological profile of selective ERß agonists to date, are there any obvious clinical liabilities that might hinder their development? Answering this question, again, requires rigorous testing of a variety of compounds to separate observations based on mechanism from those that might be idiosyncratic. Given the widespread expression of ERß, it was surprising to us that we could find essentially no ERß-dependent genes in normal (healthy) mice dosed with estradiol (98). This observation contributed to the construction of the challenge hypothesis, i.e. that ERß’s function becomes apparent only when the animal is stressed, ill, or otherwise compromised. If this is true, then effects of ERß activation in normal tissues may not lead to many responses and will afford less opportunity for unwanted side effects.

One very difficult but critical question to answer is the influence of ERß agonists on the risk of developing breast cancer in humans. Preclinically, proliferative effects of estrogens on human breast cancer cells in vitro or in animal models of breast cancer is easily demonstrated. For example, a number of human breast cancer cell lines proliferate in response to estrogens, and antiestrogens or selective ER modulators, which are clinically useful agents to treat human cancers, block this effect. In addition, transgenic mice expressing the ER coactivator amplified in breast cancer 1/steroid receptor coactivator 3 develop mammary tumors whereas null mice are resistant (99, 100). These studies have primarily focused on the role of ER in mediating the effects of estrogens.

It should be remembered however, that, even though many estrogen-responsive preclinical models of breast cancer exist, a large-scale clinical trial involving more than 10,000 women taking 0.625 mg/d of conjugated equine estrogens for a mean of 6.8 yr demonstrated that this regimen did not increase the risk for developing breast cancer (101). In fact, there was a trend toward a decreased risk (hazard ratio with 95% confidence interval = 0.59–1.01). This observation demonstrates that the effects of estrogens seen in preclinical models of breast cancer do not wholly translate to humans.

ERß is expressed in the human mammary gland, and many studies have tried to define its role. The goal of mapping studies is to correlate expression of ER and ERß with clinical parameters. It should be cautioned, however, that a correlation between two observations does not guarantee that they are causally related. Many such studies have been conducted, and the results from those published from 1997–2003 (mRNA) or 1999–2003 (protein) are summarized in a recent review (102), and the conclusions are concordant with work published more recently (103, 104, 105, 106, 107, 108, 109). Regarding mRNA expression, the majority of these studies indicate that healthy mammary glands express more ERß mRNA than do breast cancer samples. ERß expression has been correlated with tamoxifen sensitivity and disease-free/overall survival. In breast cancer samples, ER is usually the predominant ER, and ERß expression has been reported to decrease during carcinogenesis. Promoter methylation has been implicated as a cause of reduced ERß expression (110), and a decrease in ERß expression has also been reported for other cancers such as prostate, endometrium, ovary, and colon and has led to the hypothesis that ERß is a potential tumor suppressor (see Ref. 111 for review).

A number of in vitro functional studies have been performed to examine the effect of ERß expression on the proliferation of breast cancer cells (112, 113, 114, 115, 116). Although the results are not unanimous, the majority of studies conclude that an increase in ERß expression decreases cell proliferation. Additionally, when ligand dependence of the effects was evaluated, most studies agree that the effects seen are ligand independent. Therefore, they may have minimal implication for predicting the activity of an ERß-selective agonist. Taken together, the current data do not support a role for ERß’s ligand-dependent activity in the genesis or progression of breast cancer.

	CONCLUDING REMARKS

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 
The story of ERß is a tribute to the power and possibility of genomics as a means to identify new protein family members of high scientific interest and possible clinical utility. However, although identification is relatively straightforward given today’s research and bioinformatics tools, defining function of a new protein can be seemingly slow and challenging even in a field where so much is known about its close relatives. It has taken almost 9 yr to make significant progress toward understanding the in vivo activities of ERß, and at that, we have poor understanding of the mechanisms that underlie the in vivo observations described in this review. Additionally, the degree to which ERß contributes to normal estrogen action has yet to be determined. The time needed to make these discoveries is really a reflection of the enormity and complexity of the task and the necessity of many disciplines to work together. The field of ERß research has been a remarkable partnership between public and private sector research efforts and as well between chemists, structural biologists, physiologists, and pharmacologists. With the research tools we now have available and results from ongoing clinical trials, the next few years should yield even more exciting findings and significant progress in our understanding of ERß’s function and therapeutic utility.

Note Added in Proof
Since this manuscript was accepted, data using PPT, ERB-041 and WAY-202196 have been published showing that ER stimulation, in combination with progesterone, is necessary and sufficient to elicit changes on mouse mammary gland morphology and regulation of at least one mRNA marker of hormone response (117).

	ACKNOWLEDGMENTS

 
Rick Winneker, Jim Keith, and Mark Day critically read the manuscript and provided helpful suggestions. I also thank C. Richard Lyttle for his confidence in my potential as a team leader, and for his tireless advocacy of the ERß program at Wyeth.

	FOOTNOTES

 
Disclosure: H.H. is a full-time employee of Wyeth Research.

First Published Online March 23, 2006

Abbreviations: DPN, Diarylpropionitrile; ER, estrogen receptor; ERKO, ER knockout; ßERKO_CH, mice produced in Chapel Hill; ßERKO_KI, mice established at Karolinska Institute; ßERKO_ST, mice produced at Strasbourg, France; ßERKO_WYE, mice produced at Wyeth; ERB-041, 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3-benzoxazol-5-ol; HPA, hypothalamic-pituitary-adrenal; KO, knockout; 16-LE₂, 3,17-dihydroxy-19-nor-17-pregna-1,3,5(10)-triene-21,16-lactone; PPT, propylpyrazole triol; 8ß-VE₂, 8-vinylestra-1,3,5(10)-triene-3,17ß-diol; WAY-200070, 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol; WAY-202196, 3-(3-fluoro-4-hydroxy-phenyl)-7-hydroxy-naphthonitrile.

Received for publication November 17, 2005. Accepted for publication March 16, 2006.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUMMARY OF AVAILABLE TOOLS... ER{alpha} MEDIATES THE MAJORITY... ROLES FOR ERß IN... UNANSWERED QUESTIONS ABOUT... CONCLUDING REMARKS REFERENCES

 

1. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary DNA. Science 231:1150–1154[Abstract/Free Full Text]
2. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139[CrossRef][Medline]
3. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11166[Abstract/Free Full Text]
4. Kuiper GGJM, Enmark E, Pelto Huikko M, Nilsson S, Gustafsson J A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
5. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353–365[Abstract/Free Full Text]
6. Mosselman S, Polman J, Dijkema R 1996 ER ß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
7. Kuiper G, Lemmen JG, Carlsson B, Corton JC, Safe SH, Vandersaag PT, Vanderburg P, Gustafsson JA 1998 Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139:4252–4263[Abstract/Free Full Text]
8. Kuiper GGJM, Carlsson B, Grandian K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptor and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
9. Harris HA, Bapat AR, Gonder DS, Frail DE 2002 The ligand binding profiles of estrogen receptors and ß are species dependent. Steroids 67:379–384[CrossRef][Medline]
10. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen receptor- and -ß mRNA in the rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
11. Shughure PJ, Lane MV, Scrimo PJ, Merchenthaler I 1998 Comparative distribution of estrogen receptor- (ER-) and ß (ER-ß) mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 63:498–504[CrossRef][Medline]
12. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor- (ER) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER-knockout mouse. Endocrinology 138:4613–4621[Abstract/Free Full Text]
13. Kuiper GGJM, Shughrue PJ, Merchenthaler I, Gustafsson JA 1998 The estrogen receptor ß subtype—a novel mediator of estrogen action in neuroendocrine systems. Front Neuroendocrinol 19:253–286[CrossRef][Medline]
14. Nilsson S, Kuiper G, Gustafsson JA 1998 ER ß: a novel estrogen receptor offers the potential for new drug development. Trends Endocrinol Metab 9:387–395[CrossRef]
15. Koehler KF, Helguero LA, Haldosen LA, Warner M, Gustafsson JA 2005 Reflections on the discovery and significance of estrogen receptor ß. Endocr Rev 26:465–478[Abstract/Free Full Text]
16. Hewitt SC, Harrell JC, Korach KS 2005 Lessons in estrogen biology from knockout and transgenic animals. Annu Rev Physiol 67:285–308[CrossRef][Medline]
17. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA 2001 Mechanisms of estrogen action. Physiol Rev 81:1535–1565[Abstract/Free Full Text]
18. Couse JF, Curtis SW, Washburn TF, Lindzey J, Golding TS, Lubahn DB, Smithies O, Korach KS 1995 Analysis of transcription and estrogen insensitivity in the female mouse after targeted disruption of the estrogen receptor gene. Mol Endocrinol 9:1441–1454[Abstract]
19. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M 2000 Effect of single and compound knockouts of estrogen receptors (ER ) and ß (ER ß) on mouse reproductive phenotypes. Development 127:4277–4291[Abstract]
20. Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, Sar M, Korach KS, Gustafsson JA, Smithies O 1998 Generation and reproductive phenotypes of mice lacking estrogen receptor ß. Proc Natl Acad Sci USA 95:15677–15682[Abstract/Free Full Text]
21. Shughrue PJ, Askew GR, Dellovade TL, Merchenthaler I 2002 Estrogen-binding sites and their functional capacity in estrogen receptor double knockout mouse brain. Endocrinology 143:1643–1650[Abstract/Free Full Text]
22. Veeneman GH 2005 Nonsteroidal subtype selective estrogens. Curr Med Chem 12:1077–1136[CrossRef][Medline]
23. Meyers MJ, Sun J, Carlson KE, Marriner GA, Katzenellenbogen BS, Katzenellenbogen JA 2001 Estrogen receptor-ß potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues. J Med Chem 44:4230–4251[CrossRef][Medline]
24. Malamas MS, Manas ES, McDevitt RE, Gunawan I, Xu ZB, Collini MD, Miller CP, Dinh T, Henderson RA, Keith Jr JC, Harris HA 2004 Design and synthesis of aryl diphenolic azoles as potent and selective estrogen receptor-ß ligands. J Med Chem 47:5021–5040[CrossRef][Medline]
25. Mewshaw RE, Edsall RJ, Yang CJ, Manas ES, Xu ZB, Henderson RA, Keith JC, Harris HA 2005 ER ß ligands. 3. Exploiting two binding orientations of the 2-phenylnaphthalene scaffold to achieve ER ß selectivity. J Med Chem 48:3953–3979[CrossRef][Medline]
26. Hillisch A, Peters O, Kosemund D, Muller G, Walter A, Schneider B, Reddersen G, Elger W, Fritzmeier KH 2004 Dissecting physiological roles of estrogen receptor and ß with potent selective ligands from structure-based design. Mol Endocrinol 18:1599–1609[Abstract/Free Full Text]
27. Stauffer SR, Coletta CJ, Tedesco R, Nishiguchi G, Carlson K, Sun J, Katzenellenbogen BS, Katzenellenbogen JA 2000 Pyrazole ligands: structure-affinity/activity relationships and estrogen receptor--selective agonists. J Med Chem 43:4934–4947[CrossRef][Medline]
28. Harris HA, Katzenellenbogen JA, Katzenellenbogen BS 2002 Characterization of the biological roles of the estrogen receptors, ER and ER ß, in estrogen target tissues in vivo through the use of an ER -selective ligand. Endocrinology 143:4172–4177[Abstract/Free Full Text]
29. Hegele-Hartung C, Siebel P, Peters O, Kosemund D, Muller G, Hillisch A, Walter A, Kraetzschmar J, Fritzemeier KH 2004 Impact of isotype-selective estrogen receptor agonists on ovarian function. Proc Natl Acad Sci USA 101:5129–5134[Abstract/Free Full Text]
30. Pelzer T, Jazbutyte V, Hu K, Segerer S, Nahrendorf M, Nordbeck P, Bonz AW, Muck J, Fritzemeier KH, Hegele-Hartung C, Ertl G, Neyses L 2005 The estrogen receptor- agonist 16 -LE2 inhibits cardiac hypertrophy and improves hemodynamic function in estrogen-deficient spontaneously hypertensive rats. Cardiovasc Res 67:604–612[Abstract/Free Full Text]
31. Couse JF, Korach KS 1999 Estrogen receptor null mice: What have we learned and where will they lead us? Endocr Rev 20:358–417[Abstract/Free Full Text]
32. Hewitt SC, Deroo BJ, Hansen K, Collins J, Grissom S, Afshari CA, Korach KS 2003 Estrogen receptor-dependent genomic responses in the uterus mirror the biphasic physiological response to estrogen. Mol Endocrinol 17:2070–2083[Abstract/Free Full Text]
33. Weihua Z, Saji S, Makinen S, Cheng G, Jensen EV, Warner M, Gustafsson JA 2000 Estrogen receptor (ER) ß, a modulator of ER in the uterus. Proc Natl Acad Sci USA 97:5936–5941[Abstract/Free Full Text]
34. Tekmal RR, Liu YG, Nair HB, Jones J, Perla RP, Lubahn DB, Korach KS, Kirma N 2005 Estrogen receptor is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice. J Steroid Biochem Mol Biol 95:9–15[CrossRef][Medline]
35. Forster C, Makela S, Warri A, Kietz S, Becker D, Hultenby K, Warner M, Gustafsson JA 2002 Involvement of estrogen receptor ß in terminal differentiation of mammary gland epithelium. Proc Natl Acad Sci USA 99:15578–15583[Abstract/Free Full Text]
36. Windahl SH, Andersson G, Gustafsson J-A 2002 Elucidation of estrogen receptor function in bone with the use of mouse models. Trends Endocrinol Metab 13:195–200[CrossRef][Medline]
37. Sanchez-Criado JE, de las Mulas JM, Bellido C, Tena-Sempere M, Aguilar R, Blanco A 2004 Biological role of pituitary estrogen receptors ER and ER ß on progesterone receptor expression and action and on gonadotropin and prolactin secretion in the rat. Neuroendocrinology 79:247–258[CrossRef][Medline]
38. Miller NR, Jover T, Cohen HW, Zukin RS, Etgen AM 2005 Estrogen can act via estrogen receptor and ß to protect hippocampal neurons against global ischemia-induced cell death. Endocrinology 146:3070–3079[Abstract/Free Full Text]
39. Frasor J, Barnett DH, Danes JM, Hess R, Parlow AF, Katzenellenbogen BS 2003 Response-specific and ligand dose-dependent modulation of estrogen receptor (ER) activity by ER ß in the uterus. Endocrinology 144:3159–3166[Abstract/Free Full Text]
40. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS 1994 Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med [Erratum (1995) 332:131] 331:1056–1061
41. Harris HA, Albert LM, Leathurby Y, Malamas MS, Mewshaw RE, Miller CP, Kharode YP, Marzolf J, Komm BS, Winneker RC, Frail DE, Henderson RA, Zhu Y, Keith Jr JC 2003 Evaluation of an estrogen receptor-ß agonist in animal models of human disease. Endocrinology 144:4241–4249[Abstract/Free Full Text]
42. Prins G, Birch L, Couse JF, Choi I, Katzenellenbogen BS, Korach KS 2001 Estrogen imprinting of the developing prostate gland is mediated through stromal estrogen receptor : studies with aERKO and BERKO mice. Cancer Res 61:6089–6097[Abstract/Free Full Text]
43. Omoto Y, Imamov O, Warner M, Gustafsson JA 2005 Estrogen receptor- and imprinting of the neonatal mouse ventral prostate by estrogen. Proc Natl Acad Sci USA 102:1484–1489[Abstract/Free Full Text]
44. Weihua Z, Makela S, Andersson Leif C, Salmi S, Saji S, Webster Jeanette I, Jensen Elwood V, Nilsson S, Warner M, Gustafsson J-A 2001 A role for estrogen receptor ß in the regulation of growth of the ventral prostate. Proc Natl Acad Sci USA 98:6330–6335[Abstract/Free Full Text]
45. Couse JF, Hewitt SC, Korach KS 2000 Receptor null mice reveal contrasting roles for estrogen receptor and ß in reproductive tissues. J Steroid Biochem Mol Biol 74:287–296[CrossRef][Medline]
46. Weihua Z, Lathe R, Warner M, Gustafsson JA 2002 An endocrine pathway in the prostate, ER ß, AR, 5 -androstane-3 ß,17 ß-diol, and CYP7B1, regulates prostate growth. Proc Natl Acad Sci USA 99:13589–13594[Abstract/Free Full Text]
47. Taylor RA, Cowin P, Couse JF, Korach KS, Risbridger GP 2006 17ß-Estradiol induces apoptosis in the developing rodent prostate independently of ER or ERß. Endocrinology 147:191–200[Abstract/Free Full Text]
48. Opas EE, Gentile MA, Kimmel DB, Rodan GA, Schmidt A 2006 Estrogenic control of thermoregulation ERKO and ERßKO mice. Matuitas 53:210–216
49. Mendelsohn ME, Karas RH 2005 Molecular and cellular basis of cardiovascular gender differences. Science 308:1583–1587[Abstract/Free Full Text]
50. Grodstein F, Manson JE, Stampfer MJ 2006 Hormone therapy and coronary heart disease: the role of time since menopause and age at hormone initiation. J Women’s Health 15:35–44[CrossRef]
51. Pare G, Krust A, Karas RH, Dupont S, Aronovitz M, Chambon P, Mendelsohn ME 2002 Estrogen receptor- mediates the protective effects of estrogen against vascular injury. Circ Res 90:1087–1092[Abstract/Free Full Text]
52. Zhu Y, Bian Z, Lu P, Karas RH, Bao L, Cox D, Hodgin J, Shaul PW, Thoren P, Smithies O, Gustafsson JA, Mendelsohn ME 2002 Abnormal vascular function and hypertension in mice deficient in estrogen receptor ß. Science 295:505–508[Abstract/Free Full Text]
53. Bolego C, Cignarella A, Sanvito P, Pelosi V, Pellegatta F, Puglisi L, Pinna C 2005 The acute estrogenic dilation of rat aorta is mediated solely by selective estrogen receptor- agonists and is abolished by estrogen deprivation. J Pharmacol Exp Ther 313:1203–1208[Abstract/Free Full Text]
54. Montgomery S, Shaw L, Pantelides N, Taggart M, Austin C 2003 Acute effects of oestrogen receptor subtype-specific agonists on vascular contractility. Br J Pharmacol 139:1249–1253[CrossRef][Medline]
55. Al Zubair K, Razak A, Bexis S, Docherty JR 2005 Relaxations to oestrogen receptor subtype selective agonists in rat and mouse arteries. Eur J Pharmacol 513:101–108[CrossRef][Medline]
56. Widder J, Pelzer T, von Poser-Klein C, Hu K, Jazbutyte V, Fritzemeier KH, Hegele-Hartung C, Neyses L, Bauersachs J 2003 Improvement of endothelial dysfunction by selective estrogen receptor- stimulation in ovariectomized SHR. Hypertension 42:991–996[Abstract/Free Full Text]
57. Korte T, Fuchs M, Arkudas A, Geertz S, Meyer R, Gardiwal A, Klein G, Niehaus M, Krust A, Chambon P, Drexler H, Fink K, Grohe C 2005 Female mice lacking estrogen receptor ß display prolonged ventricular repolarization and reduced ventricular automaticity after myocardial infarction. Circulation 111:2282–2290
58. Pelzer T, Loza PAA, Hu K, Bayer B, Dienesch C, Calvillo L, Couse JF, Korach KS, Neyses L, Ertl G 2005 Increased mortality and aggravation of heart failure in estrogen receptor-ß knockout mice after myocardial infarction. Circulation 111:1492–1498
59. Gabel SA, Walker VR, London RE, Steenbergen C, Korach KS, Murphy E 2005 Estrogen receptor ß mediates gender differences in ischemia/reperfusion injury. J Mol Cell Cardiol 38:289–297[CrossRef][Medline]
60. Skavdahl M, Steenbergen C, Clark J, Myers P, Demianenko T, Mao L, Rockman HA, Korach KS, Murphy E 2005 Estrogen receptor-ß mediates male-female differences in the development of pressure overload hypertrophy. Am J Physiol Heart Circ Physiol 288:H469–H476
61. Dubal DB, Zhu H, Yu J, Rau SW, Shughrue PJ, Merchenthaler I, Kindy MS, Wise PM 2001 Estrogen receptor , not ß, is a critica